This invention provides glucopyranosides conjugates of 2-(4-hydroxy-phenyl)-3-methyl-1-[4-(2-amin-1-yl-ethoxy)-benzyl]-1H-indol-5-ols which are useful as tissue elective estrogenic agents.
The use of hormone replacement therapy for bone loss prevention in post-menopausal women is well precedented. The normal protocol calls for estrogen supplementation using such formulations containing estrone, estriol, ethynyl estradiol or conjugated estrogens isolated from natural sources (i.e., PREMARIN; conjugated equine estrogens). In some patients, therapy may be contraindicated due to the proliferative effects of unopposed estrogens (estrogens not given in combination with progestins) have on uterine tissue. This proliferation is associated with increased risk for endometriosis and/or endometrial cancer. The effects of unopposed estrogens on breast tissue are less clear, but are of some concern. The need for estrogens which can maintain the bone sparing effect while minimizing the proliferative effects in the uterus and breast is evident. Certain nonsteroidal antiestrogens have been shown to maintain bone mass in the ovariectomized rat model as well as in human clinical trials. Tamoxifen (sold as NOVALDEX, tamoxifen citrate), for example, is a useful palliative for the treatment of breast cancer and has been demonstrated to exert an estrogen agonist-like effect on the bone, in humans. However, it is also a partial agonist in the uterus and this is cause for some concern. EVISTA (raloxifene), a benzothiophene antiestrogen, has been shown to stimulate uterine growth in the ovariectomized rat to a lesser extent than Tamoxifen while maintaining the ability to spare bone. A useful review of tissue selective estrogens is seen in the article xe2x80x9cTissue-Selective Actions Of Estrogen Analogsxe2x80x9d, Bone Vol. 17, No. 4, October 1995, 181S-190S.
The use of indoles as estrogen antagonists has been reported by Von Angerer, See, J. Med. Chem. 1990, 33, 2635-2640; J. Med. Chem. 1987, 30, 131-136. Also see Ger. Offen., DE 3821148 A1 891228 and WO 96/03375.
WO A 95 17383 (Kar Bio AB) describes indole antiestrogens with long straight chains. Another related patent WO A 93 10741 describes 5-hydroxyindole with a generic descriptor incorporating other side chains. WO 93/23374 (Otsuka Pharmaceuticals, Japan) describes compounds which differ from the present invention; where OR2 in the present formula I, below, is defined as thioalkyl and the reference discloses no such compounds having chains from the indole nitrogen having the same structure as the ones provided by the present invention. Where the side chain claimed is similar to that described herein, the compounds are amides: Acylated indoles are not claimed in the present invention. Glucuronic acid conjugates of the selective estrogen receptor modulator raloxifene (a benzothiophene) have been reported (EP 683170 A1 951122).
This invention provides compounds of Formula I having the structure 
wherein:
R1 and R2 are independently, hydrogen, alkyl chain of 1-6 carbon atoms, benzyl, acyl of 2-7 carbon atoms, benzoyl, 
X is hydrogen, alkyl of 1-6 carbon atoms, CN, halogen, trifluoromethyl, or thioalkyl of 1-6 carbon atoms;
n=1-3;
with the proviso that at least one of R1 or R2 are not hydrogen, alkyl chain of 1-6 carbon atoms, benzyl, acyl of 2-7 carbon atoms, or benzoyl;
or a pharmaceutically acceptable salt thereof which are useful as tissue selective estrogens.
The alkyl moiety of the phenol ether substituent include both straight chain as well as branched moieties. Halogen means bromine, chlorine, fluorine, and iodine.
The pharmaceutically acceptable salts include salts formed from the addition reaction with either inorganic or organic acids. Inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and nitric acid are useful. Organic acids such as acetic acid, propionic acid, citric acid, maleic acid, malic acid, tartaric acid, phthalic acid, succinic acid, methanesulfonic acid, toluenesulfonic acid, napthalenesulfonic acid, camphorsulfonic acid and benzenesulfonic acid are also useful. The pharmaceutically acceptable salts of this invention also include quaternary ammonium salts which can be prepared by quaternizing the basic amine of the compounds of this invention with an electrophilic organic halide, mesylate, tosylate, and the like. Compounds of this invention which contain a carboxylic acid may form pharmaceutically acceptable base addition salts by treating the neutral starting material with a suitable inorganic base such as hydoxides or carbonates of alkali metals such as lithium, sodium, potassium, cesium, magnesium, calcium or barium. Or, the acid may be treated with an organic base (such as various organic primary (including ammonia), secondary or tertiary amines) to form the ammonium salts.
The compounds of this invention can be synthesized according to the generic methods shown in Scheme 1. The orthogonally protected indole is formed by a modified Bischler protocol wherein the xcex1-bromo-4-pivaloyl protected hydroxypropiophenone or acetophenone 1 is reacted with 4-benzyloxyaniline in DMF in the presence of triethylamine. The reaction is monitored by TLC for consumption of starting materials. The aniline substituted material does not need to be isolated but instead, in the same flask, treated with an additional 1.25-1.5 equivalents of 4-benzyloxyaniline hydrochloride and heated to 120-160xc2x0 C. until the previous intermediate is completely consumed. The protected indole 2 is subsequently treated with a suitable base such as sodium hydride in DMF and then reacted with an appropriate benzyl chloride of type 3. The indole 4 can then be mono-deprotected by either hydrogenating the benzyl group off at the 5-position of the indole or hydrolyzing the pivaloyl group at the 4xe2x80x2-position of the 2-phenyl group of the indole. The group removed is determined by the position of the glucuronic acid conjugate desired. The removal of the benzyl group or the pivaloyl ester renders compounds of either type 5 or 6, respectively. Reaction of compound 5 or 6 with the trichloroacetimidate CAS# [150607-95-7] of the protected glucopyranoside in the presence of BF3 etherate in a polar aprotic solvent such as dichloromethane results in the glucopyranisodated compounds 7 or 8. We have found that the reaction of either 7 or 8 with the trichloroacetimidate supra in CH2Cl2 using 3 xc3x85 molecular sieves allowed for the synthesis of the glucopyranosides in very good yield. The compounds are exclusively formed as the xcex2-glucopyranosides (equatorial substituted). The pivaloylated compound 7 can be then treated with LiOH in THF/H2O/MeOH (or dioxane/MeOH/H2O) to effect the complete deprotection of the compound, to give after workup, the monoglucuronic acid 9. The mono-benzyl ether 8 can be hydrogenated by hydrogen transfer between cyclohexadiene and Pd/C and then hydrolyzed by LiOH in THF/H2O/MeOH (or dioxane/MeOH/H2O) to yield the mono glucuronic acid 10. More preferable for larger scale hydrogenations are the conditions using a 10% Pd/C catalyst, H2 and a solvent system consisting of THF/EtOH. 
Compounds wherein both phenols are glucuronidated can be prepared according to Scheme 2. The bis-benzyl protected indole 12 is prepared by a modified Bischler reaction analogously to that described for indole 2 in scheme 1. The indole can then be subsequently alkylated with the side chain 3 (same as shown in scheme 1). The substituted indole 13 can then be deprotected by hydrogenation of the benzyl ethers to form the deprotected compound 14. The deprotected compound is then bis-glucuronidated by treatment of the free phenolic containing compound with the trichloroacetimidate of the protected glucopyranoside CAS# [150607-95-7] (same reagent used as in scheme 1) to afford 15. The final deprotected bis-glucuronic acid is obtained by base hydrolysis of precursor 15 to afford product 16. The side chains can be prepared in the general fashion shown in Scheme 3. 
Estrogen antagonist activity was shown for representative compounds of this invention in a standard pharmacological test procedure using MCF-7 cells transfected with an estrogen receptor. When administered orally, the compounds of this invention act at least partially as prodrugs for the corresponding hydroxylated compounds disclosed in EP 802183. Accordingly, the compounds of this invention are tissue selective estrogens, meaning that in certain tissue containing estrogen receptors, the compounds will act as estrogen agonists, and in other tissue containing estrogen receptors, the compounds will act as antagonists. The procedure used to demonstrate the estrogen antagonist activity in MCF-7 carcinoma cells is briefly summarized below, and the results obtained are provided in Table 1.
Cell Preparation: MCF-7 cells were passaged twice a week with growth medium [D-MEM/F-12 medium containing 10% (v/v) heat-inactivated fetal bovine serum, 1% (v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1]. The cells were maintained in vented flasks at 37xc2x0 C. inside a 5% C)2/95% humidified air incubator. One day prior to treatment, the cells were plated with growth medium at 25,000/well into 96 well plates and incubated at 37xc2x0 C. overnight.
Test Procedure Conditions: The cells were infected for 2 h at 37xc2x0 C. with 50 uL/well of a 1:10 dilution of adenovirus 5-ERE-tk-luciferase in experimental medium [phenol red-free D-MEM/F-12 medium containing 10% (v/v) heat-inactivated charcoal-stripped fetal bovine serum, 1%(v/v) Penicillin-Streptomycin, 2 mM gluta-Max-1, 1 mM sodium pyruvate]. The wells were washed once with 150 uL of experimental medium. Finally, the cells were treated for 24 h at 37xc2x0 C. in replicates of 8 wells/treatment with 150 uL/well of vehicle ( less than  or equal to 0.1% v/v DMSO) or compound that was diluted  greater than  or equal to 1000 fold into experimental medium.
Dose response experiments were performed in either the agonist or antagonist modes on active compounds in log increases from 10xe2x88x9214 to 10xe2x88x925 M. From these dose-response curves, EC50 and IC50 values, respectively, were generated. The final well in each treatment group contains 5 uL of 3xc3x9710xe2x88x925 ICI-182,780 (10xe2x88x926 M final concentration) as an ER antagonist control.
After treatment the cells were lysed on a shaker for 15 min with 25 uL/well of 1xc3x97 cell culture lysis reagent (Promega Corporation). The cell lysates (20 uL) were transferred to a 96 well luminometer plate, and luciferase activity was measured in a MicroLumat LB 96 P luminometer (EG and G Bethold) using 100 uL/well of luciferase substrate (Promega Corporation). Prior to the injection of the substrate, a 1 second background measurement was made for each well. Following the injection of the substrate, luciferase activity was measured for 10 seconds after a 1 second delay. The data were transferred from the luminometer to a Macintosh personal computer and analyzed using the JMP software (SAS institute); this program subtracts the background reading from the luciferase measurement for each well and then determine the mean and standard deviation of each treatment.
Analysis of Results: The luciferase data were transformed by logarithms, and the Huber M-estimator was used to down-weight the outlying transformed observations. The JMP software was used to analyze the transformed and weighted data for one-way ANOVA (Dunnet""s test). The compound treatments were compared to the vehicle control results (0.1 nM 17xcex2-estradiol) in the antagonist mode. For the initial single dose experiment, if the compound treatment results were significantly different from the appropriate control (p less than 0.05), then the results were reported as the percent relative to the 17xcex2-estradiol control [i.e., ((compound-vehicle control)/(17xcex2-estradiol controlxe2x80x94vehicle control))xc3x97100]. The JMP software was also used to determine the EC50 and/or IC50 values from the non-linear dose-response curves.
The mono-glucuronic acid conjugates were tested in the MCF-7 assay in both the agonist as well as the antagonist (co-dosed with 10 pM 17xcex2-estradiol) modes. All four compounds tested showed antiestrogenic activity in this cell system and none of the compounds showed significant agonist activity in these cells. This is a desirable outcome since the MCF-7 cell line is derived from human mammary tissue and these results indicate that the compounds were counteracting the proliferative effects of estrogen activity in these cells. This also is a positive indication in that these compounds are showing cell permeability and receptor binding affinity (since the MCF-7 cells express estrogen receptors). The data for the compounds is shown in Table 1 infra.
As stated above, the compounds of this invention are tissue selective estrogens: acting as estrogen agonists on certain tissue, and antagonists on other tissue. In particular, the compounds of this invention act as estrogen receptor agonists in providing protection against osteoporisis, lowering lipid levels, and increasing HDL levels. The compounds of this invention act as estrogen receptor antagonists in inhibiting uterine growth (as a potential side effect from the administration of estrogenic compounds), provide protection against breast cancer, provide contraception, inhibit dementias, and provide cognition enhancement.
Accordingly, the compounds of this invention are useful in treating certain conditions resulting from estrogen deficiency, such as bone loss or osteoporosis which may have resulted from an imbalance in an individual""s formation of new bone tissues and the resorption of older tissues, leading to a net loss of bone. Bone depletion results in a range of individuals, particularly in post-menopausal women, women who have undergone bilateral oophorectomy, those receiving or who have received extended corticosteroid therapies, those experiencing gonadal dysgenesis, and those suffering from Cushing""s syndrome. These compounds may also address individuals with special needs for bone, including teeth and oral bone, replacement, bone fractures, defective bone structures, individuals having bone-related surgeries and/or the implantation of prosthesis. In addition to those problems described above, these compounds can be used in treatments for osteoarthritis, hypocalcemia, hypercalcemia, Paget""s disease, osteomalacia, osteohalisteresis, multiple myeloma and other forms of cancer having deleterious effects on bone tissues. Other conditions resulting from estrogen deficiency which can be treated with compounds of this invention include prostatic hypertrophy, vaginal and skin atrophy, acne, cardiovascular disease, contraception in pre-menopausal women, as well as hormone replacement therapy in post-menopausal women or in other estrogen deficiency states where estrogen supplementation would be beneficial.
As the compounds of this invention also act as estrogen antagonists in certain tissue, they are useful in providing antiestrogen therapy, particularly in treating male pattern baldness, dysfunctional uterine bleeding, endometrial polyps, benign breast disease, uterine leiomyomas, adenomyosis, in treating neoplasms such as ovarian cancer, breast cancer, endometrial cancer, melanoma, prostrate cancer, cancers of the colon, and CNS cancers, in treating endometriosis, polycystic ovary syndrome, infertility Alzheimer""s disease, cognitive decline and other CNS disorders, and in providing contraception. The compounds of this invention are also useful in treating disease states where amenorrhea is advantageous, such as leukemia, endometrial ablations, chronic renal or hepatic disease or coagulation diseases or disorders.
Additionally, compounds 27, 28, 33, and 34 are intermediates useful in the preparation of compounds 29, 30, 35, 36, 37, and 38 as shown in Schemes 5 and 6 (below).
Effective administration of these compounds may be given at a dose of from about 0.1 mg/day to about 1,000 mg/day. Preferably, administration will be from about 10 mg/day to about 600 mg/day, more preferably from about 50 mg/day to about 600 mg/day, in a single dose or in two or more divided doses. Such doses may be administered in any manner useful in directing the active compounds herein to the recipient""s bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, vaginally, and transdermally. For the purposes of this disclosure, transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the present compounds, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).
Oral formulations containing the active compounds of this invention may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, as suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g. corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, , xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium: sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. Oral formulations herein may utilize standard delay or time release formulations to alter the absorption of the active compound(s). Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository""s melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.
It is understood that the dosage, regimen and mode of administration of these compounds will vary according to the malady, the individual being treated, and subject to the judgement of the medical practitioner involved. It is preferred that the administration of one or more of the compounds herein begin at a low dose and be increased until the desired effects are achieved.
The following procedures describe the preparation of representative examples of this invention.
All reactions were carried out under a nitrogen atmosphere. Chromatography was performed using 230-400 mesh silica gel. Thin layer chromatography was performed with silica gel plates. 1H NMR spectra were obtained on a Bruker AM-400, GE QE 300, Bruker DPX-300 or DPX-301 instrument and chemical shifts reported in ppm. Melting points were determined on a Thomas-Hoover apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer diffraction grating, Mattson 5020FT-IR or Perkin-Elmer 784 spectrophotometers. Mass spectra were recorded on a Kratos MS 50 or Finnigan 8230 mass spectrometers. LC/MS were preformed on a Micromass system, Model Platform LC with a HP 1100 LC system and a Diode Array detector. The column used was a 2.0xc3x9750 mm C18 3 xcexcm column. The mobile phase used were as followed: A=950 (10 mM NH4OAc): 50 (CH3CN); B=50 (10 mM NH4OAc):950 (CH3CN). The gradient used is as followed, t=0, 100% A, 0% B; t=15, 0% A, 100% B. BPLC were recorded on a Waters 60F Pump HPLC system with a 4.6 mmxc3x9715 cm LUNA 3 xcexcm, C18 column and a 996 Diode Array detector (at 300 nm or 220 nm). Flow=1.0 mL/min. Temp=30xc2x0 C. The mobile phase used were as followed: A=950 H2O:50 CH3CN, 20 mM K2HPO4/H3PO4; B=300 H2O:700 CH3CN, 20 mM K2HPO4/H3PO4. The gradient used is as followed, t=0, 100% B, 0% C; t=55, 0% B, 100% C. Elemental analyses were obtained with a Perkin-Elmer 2400 elemental analyzer. Compounds for which CHN are reported are within 0.4% of the theoretical value for the formula given unless expressed otherwise.